Hypertension is a major risk factor for brain, heart, and kidney disease. Although its pathogenesis remains largely unknown, known genetic causes of hypertension involve mutations that disrupt renal salt regulation. Our long-term goal is to elucidate how dietary K intake affects gene/protein expression in the kidney, and through these mechanisms, modulate salt balance. There are two types of apical K+ channels mediate this K+ recycling: a 30 pS small- conductance (SK) channel and a 70 pS intermediate-conductance (IK) channel. We hypothesize that "the IK channel is a multi-subunit channel made up of SK plus one or more additional subunits". ROMK forms the 30 pS K+ recycling/secretory channel in the thick ascending limb (TAL) and collecting duct and mediates apical K recycling in TAL and net K+ secretion by aldosterone-sensitive distal nephron cells in the connecting tubule and cortical collecting duct of the kidney. ROMK knockout mice have shown that ROMK is also required for the formation of the apical intermediate-conductance 70 pS channel in TAL. The 70 pS K+ channel contributes importantly to apical K+ recycling in TAL and is up-regulated during adaptation of a high K diet. The overall goal of our proposal is to identify the protein(s) that, together with ROMK, form the intermediate- conductance (IK) 70 pS K channel in TAL. The specific goals are to: 1) identify the protein(s) that coassemble with ROMK subunits to form an IK channel by using standard expression screening techniques in Xenopus laevis oocytes. 2) characterize the protein(s) that coassemble with ROMK subunits to form an IK using patch clamping of Xenopus laevis oocytes and molecular biology. 3) determine the molecular mechanisms of IK regulation using molecular structure/function and biophysical approaches. Overall, this research will elucidate the regulatory mechanisms controlling renal salt absorption by TAL, a critical function required for normal salt homeostasis. This new heteromeric K+ channel would provide a potential target for the design of a TAL diuretic that would provide a new treatment option for renal disease and hypertension. PUBLIC HEALTH RELEVANCE Loss-of-function mutations in the human ROMK gene (Kcnj1) cause Type II Bartter's syndrome. This proposal is to identify the related protein(s) that together with ROMK form the intermediate- conductance 70 pS K channels which plays a vital role in dietary physiology. This study will provide very important information to elucidate the regulatory mechanisms controlling salt balance in kidney, further uncovering the key mechanism of hypertension.